Nana Afia Twumasi-Ankrah

Transcription

1 Nana Afia Twumasi-Ankrah Research habit of the mind- Great 10^31 analogy!! Research habits of the mind - for pushing to find a phage and setting up a bunch of enrichments Research habits of the mind - for a great notebook entry Research habits of the mind- using the lab manual Phage name Unknown (at the moment) Soil Sample Collection GPS: ( , ) 1700 Dock Street Richmond, VA, Physical Description: Soil was knocked off of an awning that collects dirt and water from an air conditioning unit at the back entrance of Bottoms Up Pizza. The awning is black and a sealed cloth, water could have traces of freon from ac unit. Awning gets sun from noon till sundown. It was a sunny and mild day. Air Temperature: C Collection Date: August 28, 2017 Discovery method Enrichment Plating choose: Direct or Enrichment plating Identification of plaque Date: Appearance: Purification of plaque (Number and method used) Describe final plaque morphology (Clear/turbid/halo/comet/size/multipl e, etc.) Conditions for production of web plate Collection of high-titer lysate Titer: Date: DNA purification, how many columns did you use? DNA quantification, Concentration/volume: Concentration: Estimated volume collected: How is your tube labeled? Restriction digest, which enzymes cut? EM Visualization Head Diameter: Tail Length: Submitted Archive Report to bacillus.phagesdb.org or Insert link here! phagesdb.org (Microbacterium phages)

2 Submitted lysate archive to Dr. Johnson How is your tube labeled? Journal August 31, 2017 Spot Test Today was my first day in the lab. Due to this fact, I missed much of the initial enrichment process (i.e. incubating soil sample 1-2 days in advance). However, with the help of others I was quickly introduced into the course. Today, I learned what it means to work "aseptically." I tried this technique when I was asked to prepare a petri dish. During this process, a Bunsen burner was lit and I was asked to work as close to the flame s heat as possible. When preparing the petri dish, I poured some top agar into a 15-milliliter conical tube filled with a small amount of microbateria foliorum, making sure to heat (sterilize) the rim of agar jar before and after I poured. Afterwards, I poured the agar and bacteria mixture into a labeled petri dish and left it to solidify. Once solidified, I began preparing for a spot test. Using a filtered phage sample, I obtained 10 microliters of the phage sample and placed a "spot" of it into the dish. My spot test was done in quadrant six on plate four. Next class we will be looking over our spot tests. If the test is successful there will be a cluster of phage growth where a bacterial lawn is not visible. If not, the process will begin again. 9/7/17 September 5th 2017 Purification My spot test was successful (phage spot shown in figure below). In order to isolate my phage I performed a serial dilution. The dilution included using five microcentrifuge tubes and filling each with 90 microliters of phage buffer solution. The tubes were labeled according to their phage concentration starting from 10-1 to 10-5 which represents the approximate concentration of the phage per tube. After the phage buffer was added, 10 microliters of my original phage sample was added to the first microcentrifuge tube (10-1), then 10 microliters of this mixture was taken and added to the second microcentrifuge tube (10-2). This process was continued until the final microcentrifuge tube (10-5). After the dilution was done I began plating. Six different petri dishes were used for plating. With each dish corresponding with an individual phage concentration starting with 100 (original phage sample) and its following dilutions. Prior to the plating, 10 microliters of each diluted phage sample ( ) was added to individual culture tubes of Microbacteria foliorum. Top Agar was then added to each tube and the combined agar mixture was added to a labeled petri dish. The agar was left for minutes to solidify. Following this, the plates were taped together and incubated overnight. Next class we will be looking at the plates to find individual phage samples. 9/7/17 September 7th 2017 Re-Purification

3 The plates from the first purification were contaminated (shown above). As a result, I had to repeat the process. First, five microcentrifuge tubes were labeled from ( ) and filled with 90 microliters of phage buffer. Then, 10 microliters of the original liquid phage sample (100) was added to the microcentrifuge tube labeled 10-1, then 10 microliters of the resulting mixture in 10-1 was added to the microcentrifuge tube labeled This cycle was continued until the final microcentrifuge tube (10-5). Afterwards, a single petri dish was prepared by adding Top Agar to a culture tube of Microbacteria foliorum. Before the agar mixture was poured into the dish, the petri dish was divided into six sections and each section was labeled from Once the dish was labeled, the agar was poured and left for 20 minutes to solidify. After it had solidified, 10 microliters of each dilution ( ) was spotted into its corresponding dish section. Next class, we will look over these spot tests and attempt to identify individual phages from each. 9/7/17 September 12, 2017 Enrichment The plates from Thursday were contaminated. To remove the unknown sources of error, a new soil sample was used. To prepare the soil sample, it needed to be enriched. First, milliliters of Bacillus thuringienisis 350 media were added to a 15-milliliter conical tube. Afterwards, the soil sample was added to the media, and the combined volume was recorded to be milliliters. Afterwards the mixture was vortexed (mixed together) and set inside a tray to be incubated overnight. Next class, we will attempt to collect phage samples from our soil mixtures. 9/12/17 September 14, 2017 Phage sample Collection A phage sample was collected from my incubated soil-bacillus mixture. Using a pipette, one milliliter of supernatant from the soil sample was taken and put inside a microcentrifuge tube. Next class, we will be looking over spot tests for plaques and other evidence of phage. Rahul Warrier 9/14/17 September 19, 2017 Re-spot and Re-enrich

4 My spot test was unsuccessful so I had to redo the enrichment process once again as well as re-spot a new phage sample. Using two different soil samples labeled one and two, four different enrichment tubes were set up. One tube was made using Gordonia terrae, two others were made using Microbacteria foliorum, and the last was made using bacillus thuringiensis (set up shown above). Each tube was filled with 9 milliliters of media and 150 microliters of each bacteria. Lastly, approximately 3 milliliters of soil sample was added to each. For the enrichment tubes using G ordonia terrae and Microbacteria foliorum the media used was PYCA. For Bacillus thuringiensis the media used was TSB. Next class I am going to collect phage samples from each enrichment tube as well as start setting up spot tests for each. 9/19/17 September 26, 2017 New Spot Test Today we tried a new method of spot testing. Using phage directly collected from my first spot test (microbacteria foliorum - August 31, 2017), a new serial dilution and spot test was made. In order to remove the phage from the plate, a pipette tip was used to scrape plaque spots. Afterwards, the tip was added to 100 microliters of phage buffer that was pipetted into a microcentrifuge tube (rinsed off into tube). This tube made up the 100 concentration. Afterwards, 10 microliters of this mixture was taken and added to another microcentrifuge tube filled with 90 microliters of phage buffer (10-1 concentration). This process was continued (described fully above) until a 10-5 microcentrifuge tube was created. These dilutions were spotted into a petri dish that was divided into six sections. Next class, we will be looking over these spot test in hopes of finally isolating a phage. Kaivalya Dandamudi 9/26/2017 September 28, 2017 Purification: Picking a Plaque

5 The serial dilution tests from the class before yielded visible phage populations from the 100 to 10-3 concentrations (shown above). The phage plaques seen were extremely small (<1 millimeter in diameter). They were circular in shape, and relatively cloudy in color. Today, I picked a single plaque from the serial dilution dish I made yesterday. Afterwards, I added this phage sample to 100 microliters of phage buffer, and did a serial dilution with it (the same process as described yesterday). Afterwards, 250 microliters of Microbacteria culture was added to each diluted sample. Once the bacteria was added, PYCA Top Agar was then added to this mixture and all the dilutions were plated in individual Petri dishes. Next class, we will look over these dishes to identify a clearly isolated phage plaque. Brenna October 3, 2017 Re-Purification (Part III) Figure 1 Figure 2 My purification yielded really strange results. The quality of the bacteria lawns in each dish was questionable (Figure 2), and while there were some small clearings (potential plaques) in these lawns they were much too cloudy and irregularly shaped to be used (Figure 1). Consequently, today was spent re-plating new serial dilutions (described before) from the original 100 concentration. After the serial dilution was done, 10 microliters of each diluted sample ( ) was added 250 microliters of Microbacteria foliorum, and the combined mixture was left to incubate for 20 minutes on the lab table. Afterwards, PYCA Top agar was added to each test tube, and the resulting mixes were quickly poured into its respective dishes. Next class, hopefully the diluted samples will be able to yield viable phage so I can move on to the next step. 10/3/17 October 5, 2017 Picking Another Plaque

6 Figure 1 Figure 2 Figure 3 The purification from yesterday was successful and I was able to see and pick a good plaque from my 10-2 dish (shown above). However, my other dishes showed the presence of various phage morphologies. On plate 10-1 a small distribution of small (< 1mm), circular, and turbid plaques are seen (Figure 1). However, on the 10-3 plate larger, more irregularly shaped, and clear plaques are shown (Figure 2). When picking a plaque to continue purifying, I considered how well the plaque was isolated from others. The plaque I chose can be found on my 10-2 dish, it has a "halo" like shape, it is approximately 2 mm in diameter (Figure 3), and relatively cloudy in appearance. After this plaque was picked, I started another serial dilution (described before) with the concentrations Then, I added 10 microliters of each diluted sample to 250 microliters of Microbacteria foliorum. This mixture was then left for 20 minutes, PYCA Top Agar was added and the resulting combination was added to labeled dishes. Next class, I will be looking over these plates. 10/5/17 October 10, 2017 Contamination and Re-Purification (Part IV) Last week's purification was unsucessful. The plates showed evidence of contamination, as a result no phage could be observed. Today was a repeat of last class in which 10 microliters of a 100 sample was diluted 3 times over ( ), incubated 20 minutes with 250 microliters of Micr obacteria foliorum, then plated with PYCA Top Agar. Next class, I will look over these plates in order to determine whether I can move on the the next stage of purification. Rahul Warrier October 12, 2017

7 Spot Testing and Re-Purification (Part V) Last week's purification was also unsucessful. This class, I decided to start again by re-picking a plaque from my original Microbacteria foliorum s pot test, diluting it, and then creating a spot test. To start, I picked a plaque from 10-1 quadrant on my original Microbacteria plate. The plaque sample was serially diluted from 100 to 10-5 (explained above). In order to both limit and detect the source of error in my trials, three different Microbacteria cultures (named October 3, October 11, and September 24). Approximately 3 mililiters of PYCA Top Agar was added to these cultures and the resulting mixtures were poured into labeled plates. After the agar had solified in the plates, a spot test was done where 10 microliters of each dilution was pipetted into the labeled (respective to concentration) quadrants of the plates. Rahul Warrier October 17, 2017 Purification Continued (Part VI) Last week's purification yielded interesting results from the October 3rd and 11th Microbacteria foliorum cultures. However, clear evidence of phage could be seen on the September 24th spot (circled in image). As a result, the purification process could be continued. Today, plaque from the September 24th plate (Concentration 10-1) was picked, diluted, incubated with culture for 15 minutes, then plated with 250 microliters of PYCA Top Agar. Next class, these plates will be looked at and reviewed for evidence of homologous plaque populations. AJ October 24, 2017 Final Purification?

8 The purification from last class was good (image shows plate with a concentration of 10-2). While only one plaque morphology was visible on the plate, their sizes varied between 1-2 mm in diameter. The image shown above was taken a few days earlier and feature clearer plaques than the ones seen today (the plaques were substantially more turbid than they were the day before). Today, I continued the purification process by picking a plaque from the plate, serially diluting it from a concentration of 100 to 10-2, adding 10 microliters of each dilution to 250 microliters of Mi crobateria and then incubating the mixture for around 20 minutes. Afterwards, approximately 3 mililiters of PYCA Top Agar was added to each test tube and immediately plated into three labeled petri dishes. Next class, I will be looking over these plates. If adequate plaque growth is noted, I will be able to move on to the amplification step, if not I will have to continue with purification. AJ October 26, 2017 Webbed Plate! My 10-1 plate from Tuesday was webbed, as a result I was able to skip a couple of steps in the amplification process (titer calculation and webbed plating trial and errors). Today, I "flooded" my webbed plate with 5 mililiters of phage buffer. The plate was left for an hour in order for the buffer to to fully diffuse through the Top Agar of the dish. Afterwards the a "snotty" fluid comprised of buffer and slightly dissolved Top Agar was seperated from the dish and poured into a 50 mililiter conical tube. The mix was ran through a centrifuge and the resulting supernatant was pipetted into a conical syringe then microfilitered into a 15 mililiter conical tube. 100 mililiters of supernatant was pipetted into a microcentrifuge (100). Five other microcentrifuge tubes ( ) were filled with 90 mililiters of phage buffer, and the starting with the 100 tube a serial dilution was conducted. Afterwards, six test tubes of Microbateria foliorum culture was labeled according to concentration and filled with a diluted phage sample. The resulting mix was left to incubate for around 20 minutes, then plated with PYCA Top Agar. Next class I will be looking over these plates to observe consistent plaque growth. Kaivalya Dandamudi October 31, 2017 Strange Results and Spot Tests for Titer

9 My results from last class were strange. The plates seemed to yield extremely cloudy plaque-like blobs that were unlike any other plaques I had seen before. To make sure my lysate was okay I performed a serial dilution from 100 to The spot tests will be used to perform a titer calculation which I will use to amplify my phage some more so I can begin isolating my phage s DNA from its capsid shell. Next class, I will judge both the quality of my lysate as well as perform a titer calculation for the next stage of phage isolation. Kaivalya Dandamudi November 2, 2017 Phage DNA Isolation The spot test from the previous yield strange results. In order to check the presence of phage in my lysate I performed a DNA isolation. Working asceptically and with gloves, 5 microliters of nuclease was pipetted in a 15 mililiter conical tube. Afterwards, 2 mililiters of lysate was pipetted into the same tue. The mixture was incubated in 37 C for 10 minutes. After waiting, 30 microliters of EDTA solution (used to denature the nuclease) was added to the lysate mix and mixed. Then, 2 militers of DNA cleanup resin was also also added and the resulting mix was gently rocked back and forth for 2 minutes to ensure the resin's complete mix with the lysate. After that the mixture was pipetted into a 5 mililiter syringe with a DNA clean-up column attached at the bottom. Fluid from the lysate was gently pushed out of the syringe into the trash (the DNA is attached to beads found in the column at the bottom of the syringe). (Turn off flame) Once the fluid was removed, 3 washes were done with 6 mililiters of 80 % isopr opanol each. During the washes, 6 militers of 80 % isopropanol is pipetted into a syringe and pushed through the DNA column into the trash (done 3 times). The purpose of the wash is to remove any waste that may be mixed with the DNA. The columns were unscrewed from the syringe and inserted into a microcentrifuge tube. The column and tube are then centrifuged under the parameters 10,000 rpm for 5 minutes. The resulting fluid (isopropanol) from the centrifuge is dumped out, the column is reinserted and the two are re-centrifuged at 10,000 rpm for 1 minute (the resulting fluid is also dumped). Afterwards, the column and the tube are placed into a heat block for approximately 60 seconds to evaporate any remaining fluid in the column. Once this is done, the DNA is eluted by gently pipetting 50 microliters of 90 C of sterile ddh2o. The column and tube are once again centrifuged at 10,000 rpm for 1 minute (the resulting fluid is kept!). After, 50 additional microliters of 90 C of sterile ddh2o are added and this is also centrifuged at 10,000 rpm for 1 minute. The DNA column is thrown away and water in the microcentrifuge is kept (the DNA is in the water). Next class, a DNA gel electrophoresis will be done to test the presence of phage DNA. If phage DNA is present, I will be able to move on to preparing the phage DNA for microscopy and further genomic analysis. If not, then I will have to make more webbed plates in order to increase the titer of my phage lysate. Brenna November 7th, 2017 DNA Gel Electrophoresis Today I prepared a 500 mililiters of 1% agarose by combining 50 mililiters of 10x TAE buffer with 450 mililiters of deionized water. Afterwards, the mixture was swirled then heated in the microwave for 1 minute (or until any agarose did not have any clumps in it). The mixture was left to cool for 5 minutes, then 7 microliters of Ethedium bromide was added. This mix was swirled then slowly poured into the gel tray and left for approximately 15 minutes to solidify. While waiting, a quick test for the concentration of DNA particles in out DNA sample was taken. Using a nanodrop (spectrometer) 2 microliters of DNA sample was tested and the concentrations of 19 and 4.5 nanograms per microliter were found for both DNA samples 1 and 2 respectively. Once the agarose had solidified, 20 microliters of DNA sample 1 was mixed with 3 microliters of blue dye and 7 microliters of water. The combined 20 microliter volume was then pipetted into a ridge in the gel. The gel on its tray was suspended in approximately 1,000 ml of 10% Buffer and electroquted at 100 volts. Next class we will look over the results from the electrophoresis in order to determine the presence of phage DNA. Kaivalya Dandamudi

10 November 9th, 2017 Three Plates and Electrophoresis results The gel electrophoresis for my phage sample did not show any presence of phage DNA. In order to increase the number of phage I had, I began preparing more high-titered lysate. In order to prepare the lysate, I spent today plating three 10-3 diluted plates. Three test tubes filled with 250 micoliters of Microbacteria foliorum recieved 10 microliters of a 10-3 lysate dilution. The test tubes were left to incubate for 20 minutes. After the incubation period, approximately 3 mililiters of PYCA Top Agar was added and the the resulting mixture was slowly poured into a labeled dish. Next class, hopefully all three plates will be webbed and ready to be flooded. 11/9/17 November 10th, 2017 Collecting Lysate and Spot Titer Today I flooded three webbed plates by pouring approximately 5 milliliters of phage buffer into the plates, then leaving them to soak for an hour. After an hour a large Pipette was used to siphon the lysate from each plate. The lysate was collected into a 20 milliliter conical tube. 10 milliliters of lysate was then taken and mixed with 40 microliters of nuclease and 4 milliliters of Phage precipitant. The nuclease-precipitate-lysate mix will be incubated at 37 degrees Celsius for approximately 30 minutes to produce a phage precipitate. Next class for DNA isolation. Additionally, 5 microliters of lysate dilutions were spotted onto a Microbacterium foliorum lawn. The spot dilutions will be used for a titer calculation later on. Brenna November 14th, 2017 DNA isolation and Purification

11 My spot test for a titer once again yielded strange results. However, after getting phage precipitate was found, the phage pellet was suspended in 500 microliters of ddh20. The pellet was slowly broken down and mixed into the water by slowly pipetting up and down and swirling when needed. Once the pellet was fully mixed into the water, 2 milliliters of pre-warmed (37 degrees Celsius) DNA Clean up resin was added. The mixture was gently swirled to mix fully. In order to isolate the DNA, 1.26 milliliters of the resin-phage DNA solution was pipeeted into a syringe with a DNA column attached at its end. The solution was slowly squeezed through the column and the liquid that flowed through was discarded. Afterwards the column was washed through with 2 milliliters of 80% isopropynol alcohol, placed on top of a microcebtrifuge tube and centrifuged at 13,000 rpm for 1 minute. Afterwards, 50 microliters of 90 C of sterile ddh2o are added and this is also centrifuged at 10,000 rpm for 1 minute, the resulting fluid in the microcentrifuge tube holding the column now contains the DNA. The DNA column is thrown away and water in the microcentrifuge is kept (the DNA is in the water). Next class, a DNA gel electrophoresis will be done to test the presence of phage DNA. Next class, we will be doing a DNA electrophoresis with our DNA samples. Brenna November 16th, 2017 Nano-drop and TEM sample At the beginning of class, I collected a milliliter of phage sample and pippeted it into a microcentrifuge tube. This sample will be used for the Transmission Microscope. Afterwards, we conducted a nanodrop test where my DNA elusions from the previous class were determined to be approximately 20 and 27 nanograms (of DNA) per microliter. Afterwards, approximately 7.4 microliters of the second DNA sample (27 ng/ul) 3 microliters of blue dye, and 9.6 microliters of deionized water were combined into a microcentrifuge tube. The combined solution was left for a gel electrophoresis test to be conducted before next class. Next class, i will be looking over results from the gel electrophoresis. Rahul Warrier November 21, 2017 Electron Microscopy Staining Today, I did an electron microcopy staining from my lysate sample. First, 10 microliters of lysate was applied to a copper pellet and left for 5 minutes. Any remaining lysate was wicked off, and 10 microliters of ddh20 was added to the pellet. The water was left for a minute, wicked off, then reapplied for an additional minute only to be wicked off once more. Once the pellet had underwent two washes with the water, 10 microliters of uranyl acetate was pipetted onto the pellet and left for 2 minutes. After the two minutes, the uranyl acetate was also wicked off and the stained copper pellet was placed into the slot D8 on the tray. The sample will be used for electron microscopy so a physical image of my phage can be taken. Ayshah Asmat 11/21/17 November 28, 2017 Gel Results and Webbed Plates

12 The results from gel electrophoresis test showed no evidence of phage DNA in my sample.as a result, I have to go back a few steps to create more webbed plates and collect more lysate in order to increase my DNA concentration. Today I practiced calculating the concentration of DNA needed for a restriction enzyme. One example I did was: "If given a DNA sample with a concentration of 540 nanograms per microliter and 500 nanograms are needed then how much volume of DNA is necessary?" I found the solution to this problem by setting up the following equation: 1 microliter / 540 nanograms x 500 nanograms = 500 / 540 microliters = 0.92 microliters of DNA sample needed Rahul Warrier December 5th, 2017 Lysate Archiving and "UncleBen" Today I archived my lysate by pipetting 200 microliters of my lysate into a small archiving tube with a red cap filled with red beads and glycerol-dextrose solution. Once two of these tubes were filled, each was labeled and stored. AJ